Collagen is the most abundant protein representing nearly 30% of total proteins in the animal body. It is the major component of extracellular matrices and is vital for mechanical protection of tissues, organs, and physiological regulation of cellular environment. Collagen and collagen based forms in combination with other synthetic, semi-synthetic and biopolymers have broad applications in wound healing, tissue engineering, regenerative medicine, moisturiser, food industry, laboratory reagents, health drink, etc.
Aquatic source of collagen is of great importance worldwide. Marine source of Type I collagen are fishes such as codfish [K. A. Piez, Biochemistry, 1965 December; 4(12):2590-2596], salmon, skin of Big eye snapper, Large fin long barbel catfish, Cuttlefish, scales of Lizard fish, Horse mackerel, Grey mullet, Flying fish, Yellowback seabream etc. [T. H. Silva, et. al. Mar. Drugs 2014, 12, 5881-5901], or invertebrate marine animals, such as marine sponges, star fish, octopus or jellyfish [T. Nagai, et. al., J Sci Food Agric 79:855-858; 1999]. The marine sources contains higher amount of minerals and therefore, removing of them is costly, time consuming and cumbersome and usually ethylenediaminetetraacetic acid (EDTA) is used for this. However, remnant EDTA in collagen to be used for medical devices is very harmful for skeletal tissues and can cause to decay of bones.
Further, the denaturation temperature (Td) of collagen from marine source is low. The denaturation temperature (Td) of edible jelly fish collagen is 26.0° C. [T. Nagai, J Sci Food Agric 79:855-858, 1999] and that of starfish is 24.7° C. [B. Wang, Mar. Drugs 2013, 11, 4641-4661].
The mineral content in fresh water fish scale is relatively less. Moreover, this source is free from the fat or turbid matter. The scales and other external part of fish, considered to be biological wastes, are rich sources of collagen. The extraction procedure of collagen from theses sources is simple, less time consuming and can be extracted using less chemical treatments. This collagen of fresh water origin has relatively higher denaturation temperature (Td) mainly due to high content of hydroxyproline associated with nature of their habitat.
It is well known that the extraction of collagen from fish scale usually comprises either of the enzymatic method/hot water extraction method/methanol/EDTA or a combination of them.
CN 103773828 discloses method for extraction of collagen from fish scale by conducting crushing, hot water and enzymatic treatment using 1-3% of protease assisted by ultrasonic wave. Without acid-base pre-treatment, the method provided has no pollution to the environment, can achieve good separation and purification effect, and does not affect the biological activity of collagen.
CN 1814782, mentions an enzyme engineering technology including taking scales as the main raw material, drying and crushing them to be sieved, mixed with water, enzymolyzed with proteinases then to eliminate; enzymes and filter them to get the solution to be concentrated, dried to get the product and extracting small molecular collagens from the scales by a biological enzyme cutting technology where hydrolysis is carried out at 35-70° C. for 3 to 12 hours followed by heating reaction liquid at 90° C. for constant temperature enzyme inactivation.
CN 103570827 discloses preparation methods of fish scale collagen protein comprising four methods: an acid method, alkali/salt method, an enzymatic method and a hot-water, extraction method using ultrasonic wave which improves extraction of collagen from fish scale.
US20070231878 discloses a method of protein hydrolysis using enzyme and treating in warm water to extract collagen of fish scale; followed by centrifugation of the hydrolyte; taking out the supernatant of the hydrolyte rice; and drying the supernatant to become collagen powder.
US20070231878 (pg 3 para 0047) discloses extraction of collagen using acetic acid, and the method disclosed produces 90% pure collagen.
Pati et al. has reported extraction of collagen from fresh water fish scales of Rahu and Katla with higher thermal stability and ˜5% yield by EDTA based dissolution [F. Pati, B. Adhikari, S. Dhara, Isolation and characterization of fish scale collagen of higher thermal stability, Bioresource Technology, 101, 3737-3742, 2010]. However, the collagen yield was found not of desired level and was limited to up to 5% of the scales by EDTA treatment.
CN 1888075 describes a method of extracting collagen from fish scale comprising of steps of defatting fish scale with mixture solution of methanol and distilled water, desalting with 0.075 mol/L concentration EDTA solution, enzymolysis with pepsin, centrifuging and spray drying to obtain crude product.
CN 1332981 discloses extraction method of collagen by stirring the scale with 14-fold warm water in a water vessel for 1 hour repeatedly.
CN 104004086 A also discloses pulsed ultrasound assisted hot water treatment where the temperature of the water is 40-80° C. and treatment time is 20-70 minutes.
Importantly, the concept of the modern development of dressing has undergone a fundamental change and now its function is not limited to cover the wound bed only but it should also promote tissue repair functions, provide habitat for the new organization to accelerate wound healing, reduce scarring and prevent bacterial contamination, and with its biodegradability, reduced the number of dressing change.
Chitosan, an amino-polysaccharide having resemblance with glycosaminoglycan, is not present in living bodies and apparently a foreign body to living organisms. A method of improving biocompatibility of chitosan by chemical modification or by blending with bioactive derivative is thus necessary. Composite biomaterial consisting of chitosan and collagen for artificial skin, artificial blood vessel, would-coating material, or adhesion preventive agent has been studied by many researchers (JP Patent Publication (Kokai) Nous. 56-133344 A (1981) and 63-59706 A (1988).
Alginate, a polysaccharide based polymer known for wound healing applications, could also be combined with chitosan, different organic acids and/or collagen to form hydrogel, superabsorbent materials for wound healing, external wound dressing and as cell delivery vehicles. In particular, hyaluronic acid, alginate, cellulose, chitosan and, to a lesser extent other polysaccharides, received attention for use for the development of several biomedical applications such as tissue engineering and controlled release of drugs and pharmaceutical proteins.
CN 1167471 describes the low cost biological composite dressing comprising of non-woven fabrics, chitosan, collagen I, and calcium alginate to be used in wound healing, has good biocompatibility, permeability, elasticity, flexibility and used as drug carriers, dosing dressing, improved for clinical use.
CN 1381274 discloses a composite biologic adhesive bandage composed of pressure-sensitive adhesive hydro-entangled cloth, hygroscopic pad, non-woven cloth, chitosan, collagen type I and medicine preparation.
Its advantages are high penetrability, elasticity, flexibility, bioactivity, biodegradability, and cost-effectiveness.
EP0200574 discloses a biologically compatible material comprising composite materials of N-succinyl chitosan and collagen for preparing wound dressing materials, vascular pro-artificial skins and hemostatic agents.
EP 1115432 relates to the fabrication of dermal scaffold and bio-artificial dermis using neutralized chitosan sponge, neutralized chitosan/collagen mixed sponge containing chitosan fabrics is mentioned and these are extremely useful for wound healing therapy.
However, there is still a need for a simpler, environmentally friendly process for extraction of collagen of high purity with minimum chemical treatment which makes the extracted collagen free from toxic chemicals and cost effective. In another aspect it is also necessary to work on advancements related to polyelectrolyte based bioactive super-absorbent having efficient wound healing property.